Diagnosis, prognosis, prevention and / or treatment of heterotopic ossification

Abstract

The present invention refers to a method for the diagnosis, prognosis, prevention and / or treatment of heterotopic ossification (HO). On the other hand, the present invention also refers to a method for promoting osteogenesis, promoting chondrogenesis and / or inhibiting adipogenesis.

Description

[0001] DIAGNOSIS, PROGNOSIS, PREVENTION AND / OR TREATMENT OF HETEROTOPIC OSSIFICATION FIELD OF THE INVENTION

[0002] The present invention refers to the medical field. Particularly, the present invention refers to a method for the diagnosis, prognosis, prevention and / or treatment of heterotopic ossification (HO). On the other hand, the present invention also refers to a method for promoting osteogenesis, promoting chondrogenesis and / or inhibiting adipogenesis.

[0003] STATE OF THE ART

[0004] Progressive Osseous Heteroplasia (POH) (OMIM 166350) is an ultra-rare genetic disease characterized by HO of the skin, subcutaneous fat, and skeletal muscle. POH is distinguished clinically from other related genetic disorders (Albright hereditary dystrophy (AHO), pseudohypoparathyroidism (PHP), pseudopseudohypoparathyroidism (PPHP), and primary osteoma cutis) by the deep and progressive nature of the ossifications in the absence of hormone resistance or other AHO features (i.e., short stature, obesity, round face, brachydactyly, and neurob ehavi oral problems). Most POH patients start developing ectopic bone before one year of age, which causes chronic pain and joint ankylosis, and some of them require surgery or even amputation during childhood or puberty. Nowadays, the only treatment option for these patients is the surgical resection of ossifications, which usually leads to their recurrence.

[0005] Most cases of POH are caused by heterozygous inactivating mutations in GNAS, a complex locus which encodes several transcripts with unique promoters and first exons and common downstream exons. Adding yet another level of complexity, the expression of GNAS transcripts is regulated by genomic imprinting, with some of the alternative first exons being transcribed exclusively from the maternal or paternal allele. The main product of this locus is the alpha subunit of the G-stimulatory protein of adenylyl cyclase (Gsa), which is expressed biallelically in most tissues. Gsa is ubiquitously expressed and activated by many G protein-coupled receptors (GPRCs), many of which play critical roles in bone development and remodelling. As a result of GNAS inactivation, less adenylyl cyclase is activated upon ligand binding to receptors, and thus less cyclic adenosine monophosphate (cAMP) is produced. Importantly, inhibition of cAMP signalling has been demonstrated to enhance osteoblast differentiation.Nonetheless, the presence of a specific GNAS mutation does not predict a specific disorder, phenotype, or severity of progression. For instance, the same heterozygous 4-base pair deletion has been associated with different phenotypes of AHO, PHP, PPHP, and POH. Strikingly, this specific mutation has been found in both obese PHP / PPHP patients and POH patients, who are never obese. Interestingly, ossifications in POH patients manifest in a mosaic pattern, following a dermomyotomal distribution, which has been suggested to be the consequence of loss of heterozygosity at the GNAS locus. However, even though heterozygous mutations in a mosaic state with homozygous wild-type GNAS have been identified in POH patients and asymptomatic parents, biallelic inactivation of GNAS has never been found in clinical samples.

[0006] GNAS loss-of-function mutations affect numerous signalling pathways, including key regulators of skeletal maturation and regeneration, such as Wingless-related integration site (Wnt) and Hedgehog (Hh), ultimately leading to aberrant upregulation of bone-forming genes. The development of HO results from the pathological differentiation of progenitor cells, which is promoted by inflammatory signals and growth factors such as interleukin- ip (IL-ip) and transforming growth factor pi (TGFpi). During the formation of ectopic bone, proteomic biomarkers of HO, such as alkaline phosphatase (ALPL) and osteopontin / secreted phosphoprotein- 1 (SPP1), are markedly increased. Nonetheless, the molecular mechanisms underlying the progression of this disease remain poorly understood. Likewise, the cellular origin of HO has yet to be elucidated, and whether it derives from tissue-resident or circulating progenitors remains controversial. Emerging evidence indicates that peripheral nerve-derived cells may contribute to this pathological process by differentiating into transient brown adipocyte-like cells and early chondrocytes upon bone morphogenetic protein 2 (BMP2) stimulation. Within the HO microenvironment, these brown adipocyte-like cells regulate oxygen tension, which is critical for chondrogenesis and vascularization.

[0007] There is an unmet medical need of finding reliable methods for the diagnosis, prognosis, prevention and / or treatment of HO. For instance, due to the low incidence of POH, samples from POH patients are scarce, and there are no available cell lines with the specific mutation and epigenetic context that cause this disease. With the current technology, it is not possible to reproduce this mutation while keeping the genetic, epigenetic, and tissue context of the pathology. This lack of research tools has hampered the development of newpharmacological treatments for these patients, who suffer from chronic pain and severe disability from infancy.

[0008] The present invention is focused on solving this problem and a reliable method for the diagnosis, prognosis, prevention and / or treatment of HO is herein provided. On the other hand, the present invention also provides a method for promoting osteogenesis, promoting chondrogenesis and / or inhibiting adipogenesis.

[0009] DESCRIPTION OF THE INVENTION

[0010] Brief description of the invention

[0011] The present invention refers to a method for the diagnosis, prognosis, prevention and / or treatment of HO. On the other hand, the present invention also refers to a method for promoting osteogenesis, promoting chondrogenesis and / or inhibiting adipogenesis.

[0012] Particularly, the inventors of the present invention developed two osteoblast-like cell lines from a POH patient (an ultra-rare genetic disease characterized by HO of the skin, subcutaneous fat, and skeletal muscle) with a heterozygous GNAS loss-of-function mutation. The characterization of these cell lines surprisingly revealed that GNAS wild-type allele was missing in one of them, resulting in biallelic inactivation of GNAS, and subsequent analysis of the patient’s tissues confirmed the presence of GNAS double-mutant cells (with biallelic inactivation of GNAS) in varying proportions, with up to 25% in nervous tissues. GNAS double-mutant cells exhibited an aggressive phenotype, with high expression of genes related to POH pathogenesis, even in a pro-adipogenic environment. Furthermore, GNAS doublemutant cells inhibited adipogenesis and promoted osteochondrogenesis in wild-type cells through paracrine signalling. So, the present invention describes, for the first time, the presence of GNAS double-mutant cells in a POH patient and discovered that these cells may have important implications for the severity of the pathology.

[0013] Such as it is shown in the results provided in the Examples below, the inventors of the present invention have identified GNAS double-mutant cells in ectopic bone and surrounding tissues, that biallelic inactivation of GNAS increases the expression of bone and cartilage-related genes, that GNAS double-mutant cells inhibit adipogenesis, induce osteochondrogenesis and promote inflammation through paracrine signalling, that silencing of GNAS inhibits adipogenesis and induces osteochondrogenesis, and that transcriptomic analysis of doublemutant cells reveals enrichment of genes related to bone formation and inflammation.So, the first embodiment of the present invention refers to an in vitro method for the diagnosis and / or prognosis of HO which comprises assessing the presence of cells (hereinafter cells of the invention or < / 2) characterized by biallelic inactivation of GNAS, in a biological sample obtained from the subject, particularly in bone tissue, preferably in ectopic bone tissue and / or surrounding tissues, wherein the presence of said cells is indicative that the subject may be suffering from genetic HO.

[0014] The expressions used in this patent application, such as "loss of heterozygosity (LOH) at the GNAS locus," and "GNAS double-mutant" all refer to the same fundamental concept: the inactivation of both alleles of the GNAS gene (also referred to as "biallelic inactivation of GNAS"). These terms describe scenarios where both alleles of GNAS are either mutated, deleted, or silenced, resulting in a complete loss of normal GNAS function. Within the context of this patent, these expressions are specifically associated with a2 cells, which are defined by biallelic inactivation of GNAS. The a2 cells represent the state in which both alleles of GNAS are nonfunctional, a critical mechanism in diseases like genetic heterotopic ossification. Thus, all of these terms can be considered synonymous with "biallelic inactivation of GNAS", and they are directly tied to the a2 cells, which exhibit this molecular alteration.

[0015] Detecting a loss-of-function mutation often involves identifying a mutation that disrupts the normal function of a gene. The detection of a loss-of-function mutation can be carried out by the person skilled in the art following methods which belong to the common general knowledge, for instance:

[0016] -DNA-Level Analysis:

[0017] a) Sequencing-Based Methods:

[0018] a. Sanger Sequencing: Useful for small regions of DNA or single genes. It identifies point mutations, insertions, and deletions.

[0019] b. Next-Generation Sequencing (NGS): High-throughput method to identify LoF mutations across the genome or exome, such as nonsense mutations, frameshifts, or large deletions.

[0020] c. Long-Read Sequencing: Identifies structural variants, large indels, or complex mutations that disrupt gene function.

[0021] b) PCR-Based Methods:

[0022] a. Allele-Specific PCR: Detects specific known loss-of-function mutations.b. Quantitative PCR (qPCR): Detects deletions or duplications in the gene of interest.

[0023] c) Copy Number Variation (CNV) Analysis:

[0024] a. Techniques like comparative genomic hybridization (CGH) or digital droplet PCR assess for large deletions or duplications leading to loss of gene function. -RNA-Level Analysis:

[0025] a) RT-qPCR: Measures gene expression levels; a loss-of-function mutation often results in reduced or absent mRNA.

[0026] b) RNA Sequencing (RNA-seq): Identifies aberrant splicing (e.g., exon skipping, intron retention) caused by splice-site mutations.

[0027] -Protein-Level Analysis:

[0028] a) Western Blotting: Detects absence or truncation of protein caused by a loss-of- function mutation.

[0029] b) Immunohistochemistry (IHC): Assesses protein localization and expression in tissue samples.

[0030] -Functional Assays:

[0031] a) Enzyme Activity Assays: Measure the enzymatic function of the gene product to identify functional loss.

[0032] b) Reporter Assays: Use reporter constructs to determine the functional impact of mutations on gene regulation.

[0033] c) CRISPR Screening: Introducing targeted mutations to validate loss-of-function effects in cell lines.

[0034] -Structural Prediction and In Silico Tools:

[0035] a) Pathogenicity Predictors: Tools like PolyPhen, SIFT, and MutationTaster predict the impact of variants on protein function.

[0036] b) Protein Modeling: Structural prediction to assess disruptions in protein folding or active sites.

[0037] -Phenotypic Correlation:

[0038] a) Knockout Models: Generate organism or cell models (e.g., using CRISPR) to confirm LoF through phenotypic analysis.b) Clinical Correlation: Study phenotypes in patients with identified mutations (e.g., family studies).

[0039] On the other hand, assessing whether the wild-type allele has been lost or is missing typically involves molecular biology techniques that detect or quantify the presence of specific genetic sequences. The assessment of whether a wild-type allele has been lost or is missing can be carried out by the person skilled in the art following methods which belong to the common general knowledge, for instance:

[0040] -Polymerase Chain Reaction (PCR):

[0041] a) Conventional PCR: Primers designed for the wild-type allele can indicate its presence or absence by amplifying the target sequence. A lack of amplification suggests the allele is missing.

[0042] b) Quantitative PCR (qPCR): Measures the abundance of the wild-type allele relative to a reference gene. A significant reduction in signal can indicate loss.

[0043] c) Allele-Specific PCR: Utilizes primers specific to the wild-type sequence to differentiate it from mutated or edited alleles.

[0044] -Droplet Digital PCR (ddPCR):

[0045] d) Allows precise quantification of the wild-type allele by partitioning the PCR reaction into thousands of droplets. Each droplet acts as an individual reaction chamber, increasing sensitivity for detecting loss of the allele.

[0046] -Southern Blotting:

[0047] e) Involves digesting genomic DNA, separating fragments by gel electrophoresis, transferring to a membrane, and hybridizing with a probe specific to the wild-type allele. The absence of the expected band can indicate loss.

[0048] -Next-Generation Sequencing (NGS):

[0049] f) Whole-Genome Sequencing (WGS): Identifies the presence or absence of the wildtype allele across the genome.

[0050] g) Targeted Sequencing: Focuses on the region of interest to determine if the wild-type allele is still present.

[0051] h) Copy Number Variation Analysis: Detects loss of genetic material, including the wild-type allele.-Fluorescence In Situ Hybridization (FISH):

[0052] i) Uses fluorescently labelled probes to bind specific DNA sequences in chromosomes.

[0053] If the wild-type allele is missing, the probe will fail to hybridize to that location.

[0054] -CRISPR / Cas9-Based Reporters:

[0055] j) Constructs that link Cas9 activity to a reporter gene can indicate the presence or absence of the wild-type allele when combined with specific guide RNAs targeting it. -Immunoassays:

[0056] k) If the wild-type allele encodes a protein, its loss can sometimes be inferred through assays such as Western blotting or ELISA to detect protein expression.

[0057] -Loss of Heterozygosity (LOH) Assays:

[0058] l) Detects the loss of one allele (e.g., the wild type) in heterozygous conditions by comparing the allelic balance in a sample.

[0059] -Single-Nucleotide Polymorphism (SNP) Analysis:

[0060] m) SNP genotyping can detect the presence or absence of specific alleles if the wild-type allele has a unique SNP signature.

[0061] The above-mentioned techniques may require, as a first step, isolating the cells and / or prepare a calibration curve.

[0062] So, the first embodiment is clear since the person skilled in the art could identify, with the above methods, the presence of cells characterized in that they comprise a GNAS loss-of-function mutation and lack the GNAS wild-type allele.

[0063] The second embodiment of the present invention refers to GNAS protein, gene editing tool, or gene tool for increasing the expression of GNAS gene, for use in a method for the treatment and / or prevention of HO wherein the method comprises reducing the amount or eliminating cells comprising a GNAS loss-of-function mutation and lacking the GNAS wildtype allele. Alternatively, this embodiment refers to a method for the treatment and / or prevention of HO which comprises the administration of a therapeutically effective amount of GNAS protein or the use of gene editing tool, or gene tool for increasing the expression of GNAS gene.The third embodiment of the present invention refers to a method for screening and / or producing compounds for use in a method for the treatment and / or prevention of HO which comprises assessing whether the candidate compound is able to reduce the amount or eliminate the cells comprising a GNAS loss-of-function mutation and lacking the GNAS wild-type allele, wherein if the compound is able to reduce the amount or eliminate said cells, it is an indication that the compound may be used for the treatment and / or prevention of HO. The fourth embodiment of the present invention refers to the cell line of the invention (a2) characterized by comprising a GNAS loss-of-function mutation and lacking the GNAS wildtype allele within its genetic material.

[0064] As explained above, this cell is sufficiently disclosed since the person skilled in the art could identify, with the above methods, whether the cell comprises a GNAS loss-of-function mutation and lacks the GNAS wild-type allele.

[0065] In any case, the cells of the invention (a2) have been further characterized attending to differentially upregulated and downregulated miRNAs related to bone formation and tumour progression. Particularly, miRNA sequencing of al and a2 cells (GNAS single- and doublemutant cells, respectively) identified 2,751 miRNAs, of which 422 were differentially expressed (215 upregulated and 207 downregulated) with a threshold of ±0.3 log2 fold change and a p-value cut-off of 0.05. To focus on those miRNAs with the greatest biological impact, these results were further filtered based on the number of transcripts per million (TPM), applying a cut-off of five times the median TPM for a2 transcripts for miRNAs upregulated in a2 cells and five times the median TPM for al transcripts for miRNAs downregulated in a2 cells, thus selecting the top 25% of miRNAs with the highest expression in a2 and al cells, respectively. Applying these criteria, 169 highly expressed miRNAs were upregulated in a2 cells, while 169 highly expressed miRNAs in al cells were found to be downregulated in a2 cells.

[0066] The ten most upregulated miRNAs in a2 cells (log2 fold change > 4, p < 0.0005, FDR < 0.008) included six miRNAs of the miR-302 cluster (miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302c-3p, miR-302c-5p, and miR-302d-3p). This cluster of miRNAs targets inhibitors of the BMP and TGFP signalling pathways, thereby contributing to the formation of ectopic bone. The ten most downregulated miRNAs in a2 cells (log2 fold change < -4, p < 0.0005, FDR < 0.008) comprised several miRNAs involved in tumour progression, such as miR-7156-3p and miR-135b-5p. MiR-7156-3p is negatively correlated with glioma grade,and its inhibition promotes glioma cell sternness, invasion, and growth. MiR-135b-5p reduces osteoblastoma growth and inhibits osteoblast growth and differentiation by targeting RUNX2.

[0067] The ten most upregulated and downregulated miRNAs in the cells of the invention (a2) are summarised in the Table 1 below.

[0068] Table 1. Top ten upregulated and downregulated miRNAs in a2 cells compared to al cells, as determined by microRNA sequencing.

[0069] #ID log2FC p-value FDR

[0070] hsa-miR-302a-3p 11.00 0.0000 0.0000

[0071] hsa-miR-302b-3p 10.07 0.0000 0.0000

[0072] hsa-miR-302c-3p 9.59 0.0000 0.0000

[0073] hsa-miR-3675-5p 9.39 0.0000 0.0000

[0074] ■o

[0075] « hsa-miR-302a-5p 9.39 0.0000 0.0000

[0076] g novel_miR_340 7.99 0.0000 0.0000

[0077]

[0078] hsa-miR-302c-5p 7.46 0.0000 0.0000

[0079] hsa-miR-302d-3p 7.21 0.0000 0.0000

[0080] hsa-miR-372-3p 5.69 0.0001 0.0023

[0081] hsa-miR-1269a 4.34 0.0004 0.0074

[0082] hsa-miR-6720-3p -8.75 0.0000 0.0000

[0083] hsa-miR-598-3p -8.04 0.0000 0.0000

[0084] hsa-miR-4664-3p -7.78 0.0000 0.0000

[0085] hsa-miR-1267 -6.81 0.0000 0.0000

[0086] "O

[0087] -2 hsa-miR-7156-5p -6.69 0.0000 0.0001

[0088] bi

[0089]

[0090] hsa-miR-135b-5p -5.94 0.0000 0.0000

[0091] O novel_miR_526 -5.35 0.0004 0.0070

[0092] novel_miR_160 -4.21 0.0000 0.0000

[0093] novel_miR_9 -4.15 0.0003 0.0047

[0094] hsa-miR-148a-5p -4.06 0.0000 0.0000Moreover, the cells of the invention (a2) have been further characterized attending to the proteome. The ten most upregulated proteins in a2 cells compared to al (FC > 4, p < 0.05) were eukaryotic translation initiation factor (EIF6), a-fetoprotein (AFP), copine-1 (CPNE1), poliovirus receptor (CD 155), ATP-dependent RNA helicase DDX19B, ATP-dependent DNA helicase QI (RECQL), CTP synthase 1 (CTPS1), ATP-dependent 6-phosphofructokinase (PFKP), UDP -glucose 6-dehydrogenase (UGDH), and ATP-dependent RNA helicase DDX27. The ten most downregulated proteins (FC < 0.3, p < 0.05) were receptor expressionenhancing protein 5 (REEP5), cathepsin Z (CTSZ), protein S100-A4 (S100A4), protein NDRG1, BTB / POZ domain-containing protein KCTD12, galectin-3 (LGALS3), superkiller complex protein 3 (SKIC3), cathepsin B (CTSB), protein S100-A6 (S100A6), and heat shock protein beta- 1 (HSPB1).

[0095] In a preferred embodiment of the present invention, the mutation is NM_000516.7(GNAS):c.565_568del.

[0096] In a preferred embodiment of the present invention, the HO is a GNAS-related disease selected from the group comprising: POH, Albright hereditary dystrophy (AHO), pseudohypoparathyroidism (PHP), pseudopseudohypoparathyroidism (PPHP), and / or primary osteoma cutis.

[0097] In a preferred embodiment, the present invention refers to GNAS protein for use in a method for the treatment and / or prevention of HO, wherein the method comprises administering the exogenous GNAS protein to the patient. In a preferred embodiment, the method comprises administering the exogenous GNAS protein to the patient by means of lipid vesicles, polymers and / or by injection.

[0098] In a preferred embodiment, the gene editing tool is selected from the group comprising: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALENs (Transcription Activator-Like Effector Nucleases), Zinc Finger Nucleases (ZFNs), Base Editors, Prime Editing, RNA Editing Tools, MegaTALs, or Oligonucleotide-Directed Mutagenesis (ODM).

[0099] In a preferred embodiment, the gene tool for increasing expression of GNAS is selected from the group comprising: CRISPR Activation (CRISPRa), Synthetic Transcription Factors, Viral Vectors, Plasmid Transfection, mRNA Transfection, Gene Amplification, InducibleExpression Systems, Epigenetic Modulation, Chemical Activators and / or Promoter Engineering, or self-amplifying mRNA.

[0100] The fifth embodiment of the present invention refers to a cell suspension or cell population that comprises the above defined cells or the secretome derived thereof.

[0101] Particularly, the secretome refers to a combination of molecules that are secreted by the cell. These molecules typically include proteins, lipids, nucleic acids, extracellular vesicles, and other bioactive substances that play critical roles in intercellular communication, signalling, and modulation of the extracellular matrix. The secretome is intrinsically linked to the genome of the cell, with its composition being a functional output of the genetic and epigenetic landscape, modulated by environmental cues.

[0102] In any case, the composition the a2 secretome has been analysed in the present invention. Proteomic analysis of the >100 kDa fraction of al and a2 secretomes identified a-2-macroglobulin (A2M) being the only one significantly up-regulated in a2 cells. Other pro-osteoblastogenic factors present in a2 secretome included apolipoprotein- A, COL1, periostin, vitronectin, and complement molecules C3 and C5.

[0103] The sixth embodiment of the present invention refers to a composition comprising the cell suspension or cell population of the invention, or the secretome derived thereof, for use in a method for promoting osteogenesis, for promoting chondrogenesis and / or for inhibiting adipogenesis. Alternatively, this embodiment also refers to a method for promoting osteogenesis, for promoting chondrogenesis and / or for inhibiting adipogenesis, which comprises the administration of a therapeutically effective amount of composition comprising the cell suspension or cell population of the invention, or the secretome derived thereof.

[0104] In a preferred embodiment, the composition comprising the cell suspension or cell population of the invention, or the secretome derived thereof, are used in a method for the treatment and / or prevention of a disease which may benefit from promoting the osteogenesis selected from: Osteoporosis, Osteogenesis Imperfecta (01), Fracture Healing and Bone Defects, Paget’s Disease of Bone, Osteomalacia and Rickets, Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), Osteonecrosis (Avascular Necrosis), Bone Loss from Prolonged Immobilization, Metabolic Bone Diseases, Bone Loss Due to Hormonal Changes and / or Bone Grafts and Orthopedic Surgeries; and / or for the treatment and / or prevention of diseases which may benefit from promoting chondrogenesis selected from: Osteoarthritis, rheumatoid Arthritis, traumatic cartilage injuries, intervertebral disc degeneration,chondrodysplasias, osteochondritis dissecans, avascular necrosis, temporomandibular joint disorders, congenital cartilage defects and / or post-surgical cartilage loss, and / or for the treatment and / or prevention of a disease which may benefit from inhibiting adipogenesis selected from obesity and metabolic syndrome, type 2 diabetes, cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD), cancer or inflammatory diseases.

[0105] Particularly, the present invention refers to:

[0106] An in vitro method for the diagnosis and / or prognosis of heterotopic ossification comprising identifying cells characterized by the biallelic inactivation of GNAS in bone tissue, preferably ectopic bone tissue and / or surrounding tissues, obtained from a subject, wherein the presence of said cells indicates that the subject may be suffering from heterotopic ossification.

[0107] Method for screening compounds for use in a method for the treatment and / or prevention of heterotopic ossification which comprises assessing whether the candidate compound is capable of reducing the amount or eliminate the cells characterized by the biallelic inactivation of GNAS in bone tissue (preferably ectopic bone tissue and / or surrounding tissues, wherein the reduction or elimination of said cells is indicative that the compound may be used for the treatment and / or prevention of heterotopic ossification.

[0108] Cell line, or secretome derived thereof, wherein the cell line is characterized by the biallelic inactivation of GNAS, and wherein said cell line is specific to the bone tissue (preferably ectopic bone tissue and / or surrounding tissues).

[0109] A method for selecting a treatment for heterotopic ossification, comprising determining the presence of cells with biallelic inactivation of GNAS in bone tissue (preferably ectopic bone tissue and / or surrounding tissues), wherein the presence of said cells is indicative of a specific phenotype of heterotopic ossification and can guide the selection of an appropriate therapeutic approach.

[0110] Cell suspension or cell population comprising the above defined cells, or the secretome derived thereof.

[0111] A composition comprising the above defined cell suspension or cell population, or the secretome derived thereof, for use in a method for promoting osteogenesis, for promoting chondrogenesis and / or for inhibiting adipogenesis; preferably for use in a method for the treatment and / or prevention of a disease which may benefit from promoting the osteogenesisselected from: Osteoporosis, Osteogenesis Imperfecta (01), Fracture Healing and Bone Defects, Paget’s Disease of Bone, Osteomalacia and Rickets, Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), Osteonecrosis (Avascular Necrosis), Bone Loss from Prolonged Immobilization, Metabolic Bone Diseases, Bone Loss Due to Hormonal Changes and / or Bone Grafts and Orthopedic Surgeries; and / or for the treatment and / or prevention of diseases which may benefit from promoting chondrogenesis selected from: Osteoarthritis, rheumatoid Arthritis, traumatic cartilage injuries, intervertebral disc degeneration, chondrodysplasias, osteochondritis dissecans, avascular necrosis, temporomandibular joint disorders, congenital cartilage defects and / or post-surgical cartilage loss, and / or for the treatment and / or prevention of a disease which may benefit from inhibiting adipogenesis selected from obesity and metabolic syndrome, type 2 diabetes, cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), cancer or inflammatory diseases.

[0112] For the purpose of the present invention the following terms are defined:

[0113] • The term "comprising" means including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0114] • The term "consisting of’ means including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0115] • By “therapeutically effective dose or amount” is intended an amount that, when administered as described herein, brings about a positive therapeutic response in a subject. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0116] • “Biallelic inactivation of GNAS” refers to a state in which both alleles of the GNAS gene (one inherited from the mother and the other from the father) have lost their normal function. This can occur through various mechanisms, such as mutations thatinactivate both alleles or a combination of a mutation in one allele and the loss or silencing of the remaining allele. In simple terms, GNAS is a gene that encodes the alpha subunit of the Gs protein, which plays a critical role in various cellular functions, including intracellular signaling related to protein kinase A (PKA) regulation. Biallelic inactivation means that both alleles of the GNAS gene are unable to perform their normal function in the cell, typically resulting in a loss of Gsa protein activity in the affected tissues.

[0117] • “Ectopic bone tissue and / or surrounding tissues” refers to the formation of bone in an abnormal location, outside the normal skeleton, within tissues where bone is not normally present (typically soft tissues such as skeletal muscle and other connective tissues). Surrounding tissues refers to the adjacent soft tissues around the focus of ectopic bone formation (e.g., muscle, fascia, tendons / ligaments, and subcutaneous tissue).

[0118] Description of the figures

[0119] Figure 1. (A) Primary osteoblast-like cells were isolated from ectopic bone explants and transduced with simian virus 40 large T antigen (SV40LT). SV40LT-transduced cells were subsequently transduced with human telomerase reverse transcriptase (hTERT). Two clones, designated al and a2, were isolated from the immortalized cell population. (B) Phase contrast microscopic images of al and a2 cells. (C) Number of population doublings (PDs) accumulated by al and a2 cells against days in culture. PDs were calculated as (log Nf- log Ni) / log 2 (where Nf is the final cell population, Ni is the number of cells in the inoculum and log is the natural logarithm). PDL: population doublings level. (D) GNAS mRNA and protein expression in al and a2 cells; mRNA expression data was normalized to average. (E) Calibration curve obtained with the ratio of the Delta Rn values for the wild-type (WT) and mutated (M) alleles of different proportions of al and a2 cell mixtures against the percentage of a2 cells (R squared = 0.9540). Percentage of a2-like cells (double-mutant, or only M allele) in different tissues of the patient, as determined with the previous calibration curve. HO: tissue adjacent to ectopic bone. Bar graphs represent mean ± SEM. **, p < 0.01.

[0120] Figure 2. (A) Relative mRNA expression of leukemia inhibitory factor (LIF), SRY-Related HMG Box Gene 9 (SOX9), aggrecan (ACAN), bone morphogenetic protein 2 (BMP2), chemokine (C-C motif) ligand 2 (CCL), hypoxia-inducible factor (HIF), uncoupling protein 1 (UCP1), and transforming growth factor 1 (TGFpi) in al and a2 cells, in basal conditions.Data was normalized to clone al. (B) Relative mRNA expression of Runt-related transcription factor 2 (RUNX2), osteopontin / secreted phosphoprotein- 1 (SPP1), alkaline phosphatase (ALPL), and AC AN during 21 days of osteoblastogenic differentiation. (C) Relative mRNA expression of RUNX2, SPP1, ALPL, and AC AN during 21 days of adipogenic differentiation. (D) Relative mRNA expression of RUNX2, SPP1, ALPL, AC AN, SOX9, type I collagen (COL1), type II collagen (COL2), and type X collagen (COL 10) during 21 days of chondrogenic differentiation. Data was normalized to average al and a2 expression at day 0. Graphs represent mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (clone al vs clone a2, at the same day of differentiation). #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (clone al / a2 vs. day 0 of differentiation).

[0121] Figure 3. (A) Relative mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), fatty acid-binding protein 4 (FABP4), adiponectin (ADIPOQ), perilipin 2 (PLIN2), osteopontin / secreted phosphoprotein- 1 (SPP1), and transforming growth factor P-induced (TGFBi) in C3H10T1 / 2 cells at day 0 and after 7 days of adipogenic differentiation with al and a2 cells secretomes; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) Relative mRNA expression of SRY-Related HMG Box Gene 9 (SOX9), aggrecan (AC AN), type II collagen (COL2), collagenase 3 (MMP13), SPP1, and TGFBi in ATDC5 cells at day 0 and after 7 days of chondrogenic differentiation with al and a2 cells secretomes; *, p < 0.05; **, p < 0.01. (C) Relative mRNA expression of SPP1, chemokine (C-C motif) ligand 2 (CCL), vascular cell adhesion molecule (VCAM), Runt-related transcription factor 2 (RUNX2), and SOX9 in SaOS-2 cells at day 0 and after 3 days of osteoblastogenic differentiation with al and a2 cells secretome; *, p < 0.05; **, p < 0.01 (clone al / a2 vs. day 3 of differentiation); #, p < 0.05 (al vs. a2). (D) Relative mRNA expression of SPP1, CCL2, VCAM, intercellular adhesion molecule (ICAM), interleukin- 1 beta (IL-ip), and transforming growth factor pi (TGFpi) in THP-1 monocytes after three days of culture and co-culture with al and a2 cells; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (co-culture with al / a2 vs. day 3 of culture); #, p < 0.05; ##, p < 0.01 (al vs. a2). Data was normalized to day 7 and day 3, respectively. Graphs represent mean ± SEM.

[0122] Figure 4. (A) Relative mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), fatty acid-binding protein 4 (FABP4), adiponectin (ADIPOQ), perilipin 2 (PLIN2), osteopontin / secreted phosphoprotein- 1 (SPP1), and transforming growth factor P-induced (TGFBi) in C3H10T1 / 2 cells at day 0 and after 7 days of adipogenic differentiationwith al and a2 cells secretomes and a2 secretome fractions. (B) Relative mRNA expression of SRY-Related HMG Box Gene 9 (SOX9), aggrecan (AC AN), type II collagen (COL2), collagenase 3 (MMP13), SPP1, and TGFBi in ATDC5 cells at day 0 and after 7 days of chondrogenic differentiation with al and a2 cells secretomes and a2 secretome fractions. Data was normalized to day 7. Graphs represent mean ± SEM; *, p < 0.05; **, p < 0.01.

[0123] Figure 5. (A) Relative mRNA expression of GNAS and osteopontin / secreted phosphoprotein- 1 (SPP1) in C3H10T1 / 2 after GNAS silencing (DO) and after 7 days of osteoblastogenic (OB) or adipogenic (AD) differentiation (D7). (B) Micrographs of C3H10T1 / 2 after 7 days of adipogenic differentiation following GNAS silencing, compared to control. (C) Relative mRNA expression of SRY-Related HMG Box Gene 9 (SOX9), Runt-related transcription factor 2 (RUNX2), and type II collagen (COL2) in C3H10T1 / 2 after GNAS silencing (DO) and after 7 days of adipogenic differentiation. Graphs represent mean ± SEM; *, p < 0.05; **, p < 0.01: ****, p < 0.0001.

[0124] Figure 6. GNAS protein expression in a2 cells, a2 cells transfected with samRNA negative control (NC), and a2 cells transfected with GNAS samRNA (RNA).

[0125] Detailed description of the invention

[0126] The present invention is illustrated by means of the Example set below without the intention of limiting its cope of protection.

[0127] Example 1. MATERIAL AND METHODS

[0128] Example 1.1. Cell isolation and culture

[0129] Primary osteoblast-like cells were isolated from ectopic bone explants obtained from a patient with POH who underwent surgical excision of ossified tissues. This patient was previously diagnosed with a heterozygous frameshift mutation in GNAS, consisting of a 4-base pair deletion in exon 7 (NM_000516.7(GNAS):c.565_568del). Bone explants were washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA), placed in 100-mm adherent culture dishes (Costar Corning Incorporated, Corning, NY, USA), and cultured in basal medium, consisting of Dulbecco’s modified Eagle’s medium / Ham’s F-12 (DMEM / F-12) supplemented with 10% foetal bovine serum (FBS), 4 mM L-glutamine, and 200 U / mL penicillin / streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA). At confluence, osteoblast-like cells were subcultured with trypsin-EDTA solution (Sigma-Aldrich).Example 1.2. Cell immortalization

[0130] Primary osteoblast-like cells at the first passage were transduced by spinoculation using retrovirus produced by Phoenix Amphotropic cells (ATCC CRL-3213, cpNX-A). Primary cells were transduced with simian virus 40 large T antigen (SV40LT), and SV40LT-transduced cells were subsequently transduced with human telomerase reverse transcriptase (hTERT) (Figure 1A). For retrovirus production, two plasmids were employed: pBABE-puro-SV40LT (Addgene plasmid #13970), deposited by Thomas Roberts, and pBABE-hygro-eGFP-hTERT (Addgene plasmid #28169), deposited by Kathleen Collins. Transduced cells were selected in 2.5 pg / mL puromycin (Sigma- Aldrich) or 50 pg / mL hygromycin (Thermo Fisher Scientific).

[0131] Example 1.3. Clone isolation

[0132] Transduced cells were seeded at low density on 100 mm adherent culture dishes (100 cells / dish) and left to adhere for 18 hours. After verifying the clonal origin of each colony by microscopic observation with a Leica DMil inverted microscope (Leica Microsystems, Wetzlar, Germany), colonies were left to grow until reaching a suitable size for isolation. For clone isolation, 150 pL cloning cylinders (Sigma- Aldrich) were placed over each selected colony and cells were trypsinized and subcultured in adherent culture dishes. Two clones, designated al and a2, were selected for further characterization (Figure 1A).

[0133] Example 1.4. Analysis of morphology and proliferation

[0134] The morphology of both clones was analysed by observation with a Leica DMil inverted microscope. The formula in Equation 1 was used to calculate the cumulative population doublings (PDs) of both clones. The number of PDs per day was calculated at each passage for 15 consecutive subcultures. Generation time was calculated for each cell line at each passage as the number of PDs per day. The proliferation rates of both clones were analysed by regression.

[0135] pD =log A - log Ai

[0136] log 2

[0137] Equation 1. Formula employed to calculate population doubling (PD) at each passage, where Nfis the final cell number, Ni is the initial cell number, and log is the natural logarithm.Example 1.5. Cell differentiation

[0138] For al and a2 differentiation experiments, cells were plated in 24-well plates (4.5 x io4cells / well). Six hours after seeding, basal medium was replaced with differentiation medium. Osteoblastogenic medium consisted of DMEM high glucose supplemented with 10% FBS, 4 mM L-glutamine, 100 U / mL penicillin / streptomycin (DMEM10), 10 nM dexamethasone, 5 mM P-glycerol phosphate, and 50 pg / mL ascorbic acid-2-phosphate (all from Sigma-Aldrich). Adipogenic differentiation medium comprised DMEM10, 20 nM insulin-like growth factor 1 (IGF-1) (PeproTech Inc, Cranbury, NJ, USA), 2 pM rosiglitazone, 1 pM dexamethasone, 60 pM indomethacin, 0.5 mM 3-isobutyl-l -methylxanthine (IBMX), and 10 pg / mL insulin (all from Sigma-Aldrich). Adipogenic differentiation was induced by cycles consisting of 96 hours of adipogenic differentiation medium and 72 hours of maintenance medium (DMEM10 supplemented with 10 pg / mL insulin). Chondrogenic medium was DMEM high glucose supplemented with 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, 10 ng / ml TGF-P3 (PeproTech Inc), 100 nM dexamethasone, 50 pg / mL ascorbic acid-2 -phosphate, 40 pg / mL L-proline (Sigma-Aldrich), and IX ITS+1 liquid media supplement (12521, Sigma- Aldrich). Medium was renewed every 2-3 days.

[0139] In order to investigate the effect of al and a2 paracrine signals, cell differentiation and coculture experiments with wild-type cell lines were carried out. To obtain conditioned medium from al and a2 clones, 1.5 x io5cells were seeded per well in 6-well plates and cultured for 72 hours. For adipogenesis and chondrogenesis experiments, al and a2 were cultured in basal medium, and conditioned medium was filtered through 3-kDa Centricon filter units (Sigma-Aldrich), resuspended in differentiation medium, and filtered through a 0.22 pm-pore filter. For osteoblastogenesis experiments, al and a2 were cultured in SaOS2 differentiation medium, consisting of DMEM / F-12 supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, and 50 pg / mL ascorbic acid-2-phosphate (Sigma-Aldrich). To further characterize the effect of a2 secretome in adipogenesis and chondrogenesis, its protein content was separated by successive filtration using Centricon filter units with molecular mass cut-offs of 100, 50, 30 and 3 kDa.

[0140] For adipogenesis experiments, C3H10T1 / 2 cells were seeded in 24-well plates (104cells / well) in DMEM10. Six hours after seeding, adipogenic differentiation was induced as described above. For chondrogenesis experiments, ATDC5 cells were seeded in 24-well plates (5 x IQ3cells / well) in growth medium, consisting of DMEM / F12 supplemented with5% FBS, 4 mM L-glutamine, 200 U / mL penicillin / streptomycin, 5 pg / mL transferrin, and 30 nM sodium selenite. One day after seeding, growth medium was replaced by chondrogenic medium, consisting of growth medium supplemented with 0.25 UI / mL insulin. Medium was renewed every 2-3 days. After 7 days of differentiation, cells were lysed to obtain RNA.

[0141] For osteoblastogenesis experiments, SaOS2 cells were seeded in 6-well plates (1.2 x io5cells / well) in growth medium, consisting of DMEM / F12 supplemented with 10% FBS, 2 mM L-glutamine, and 100 U / mL penicillin / streptomycin. One day after seeding, culture medium was replaced by SaOS2 differentiation medium. Cells were lysed to obtain RNA after 3 days of culture.

[0142] Example 1.6. Co-culture experiments

[0143] For co-culture with THP-1 monocytes, 104cells of al and a2 clones were seeded in 0.4 pm-pore cell culture inserts (Costar Corning Incorporated), and 1.5 x io5cells were placed per well in 24-well plates (both in basal medium). One day after seeding, the inserts containing al and a2 cells were placed over THP-1 wells. After 72 hours of co-culture, THP-1 cells were pelleted and lysed to obtain RNA.

[0144] Example 1.7. GNAS silencing

[0145] C3H10T1 / 2 cells were seeded on 24-well plates at a density of 104cells per well. The following day, culture medium was changed for DMEM / F12 supplemented with 2% FBS. After 1-hour incubation, GNAS silencing was performed using the TriFECTa DsiRNA Kit and the siLentFect™ Lipid Reagent for RNAi (Integrated DNA Technologies, Coralville, IO, USA) in Opti-MEM (Gibco, Thermo Fisher Scientific). A negative control DsiRNA was included to confirm that the transfection process did not affect normal cell function. After 6-hour incubation, the medium was discarded, and the cells were subjected to adipogenic and osteoblastic differentiation as previously described. Cells were lysed for RNA extraction after 7 days of differentiation.

[0146] Example 1.8. GNAS self-amplifying mRNA transfection

[0147] Self-amplifying mRNA (samRNA) coding for GNAS (UniProt accession number P63092) was synthesized by GenScript (Piscataway, NJ, USA). One day before transfection, a2 cells were seeded on 24-well plates at a density of 804cells per well. LipofectamineMessengerMAX (Thermo Fisher Scientific) and 0.5 ng of GNAS samRNA were mixed in Opti-MEM and added to a2 cells. Cells were incubated with the samRNA-lipid complex for 24 hours. Negative control wells were subjected to the same procedure without the GNAS samRNA.

[0148] Example 1.9. TaqMan SNP Genotyping Assay

[0149] Tissues obtained from surgical leavings were immersed in liquid nitrogen and pulverized using a Cellcrusher (Schull, Ireland). Cytiva triplePrep Kit (Thermo Fisher Scientific) was employed to obtain genomic DNA from 20 mg of pulverized tissue or 106cultured cells. DNA quantity was determined using a NanoDrop 2000 Spectrophotometer and 5 ng were mixed with Maxima Probe / ROX qPCR Master Mix and TaqMan primers and probes specifically designed for the mutation site (NM_000516.7(GNAS):c.565_568del) (all from Thermo Fisher Scientific). TaqMan SNP Genotyping Assay was carried out in a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). For each sample, the ratio between the Delta Rn values for the wild-type allele (WT) and the mutated allele (M) were calculated. In order to determine the proportion of al / a2 mosaicism in each tissue, a calibration curve was made by mixing al and a2 cells in different proportions.

[0150] Example 1.10. Gene expression analysis

[0151] Cells were lysed using TRI Reagent (Sigma-Aldrich) and RNA was purified using E.Z.N.A. total RNA kit I (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s instructions. RNA was treated with RNase-free Dnase I (Lucigen, Middleton, WI, USA) and retro-transcribed employing the High-Capacity RNA-To-cDNA (Thermo Fisher Scientific). Gene expression levels were assessed by real-time PCR using iTaq Universal SYBR Green Supermix (BioRad, Hercules, CA, USA) in combination with specific primers in a QuantStudio 3 Real-Time PCR system. Data analysis was performed using the QuantStudio 3 / 5 Real-Time PCR Software (Thermo Fisher Scientific). Relative quantification was obtained by the 2'AACtmethod with HPRT as the reference gene.

[0152] Example 1.11. Western blotting

[0153] Proteins were isolated using RIPA buffer (Sigma-Aldrich) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and quantified using BSA Pierce Bradford assay (Thermo Fisher Scientific). Protein samples (25 pg) were loaded onto a 10%sodium dodecyl sulfate polyacrylamide gel. After electrophoresis (SDS-PAGE), proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated at 4°C overnight with the primary antibodies: rabbit monoclonal anti-GNAS (ab283266; 1:500) and mouse monoclonal anti-vinculin (abl3007; 1:1000), both from Abeam (Cambridge, UK). Incubation with the secondary antibodies goat anti-rabbit (1:5,000) and goat anti-mouse (1:10,000), both from Jackson ImmunoResearch (West Grove, PA, USA), was performed for 1 hour at room temperature. Target proteins were visualized on a ChemiDoc MP Imaging System (BioRad) employing Immobilon Chemiluminescent HRP Substrate (ECL) (Millipore, Sigma-Aldrich).

[0154] Example 1.12. RNA sequencing

[0155] RNA-seq analysis was performed by the Transcriptomics Service of IDIS using the Illumina NextSeq 2000 platform. KEGG pathway enrichment analysis was performed and rendered using Pathview.

[0156] Example 1.13. Proteomic analysis

[0157] Proteomic analysis was performed by the Proteomics Service of IDIS. Micro liquid chromatography-mass spectrometry (micro-LC-MS / MS) was performed using a hybrid quadrupole TripleTOF 6600 (SCIEX, Framingham, MA, USA). Proteomes were identified by the qualitative shot-gun data-dependent acquisition (DDA) method, and protein levels were measured by the quantitative sequential window acquisition of all theoretical mass spectra (SWATH) method. A 5% false discovery rate (FDR) and p-value < 0.05 were used to filter the dataset. Enrichment analysis was performed using the ARCHS4 Tissues database in Enrichr to identify tissue-specific gene expression patterns.

[0158] Example 2. RESULTS

[0159] Example 2.1. Biallelic inactivation of GNAS in ectopic bone and surrounding tissues

[0160] Clone al displayed a fibroblast-like cell morphology characteristic of mesenchymal stromal cells and pre-osteoblasts, while clone a2 was more polygonal-shaped (Figure IB). For both clones, a continuous growth ratio was evidenced after regression analysis (R > 0.99 and p-value < 0.0001) (Figure 1C). Mean generation time of was 2.74 ± 0.32 days for clone al and 2.65 ± 0.36 days for clone a2. There were no significant differences among clones regarding proliferation rate (p-value = 0.4768). The expression of transgenes SV40LT and hTERT was evidenced in both clones. Interestingly, clone a2 showed an almost twenty-fold reduction in GNAS gene expression in comparison with clone al, and no detectable GNAS protein expression (Figure ID). TaqMan SNP Genotyping Assay revealed that clone al was heterozygous for the mutation (NM_000516.7(GNAS):c.565_568del), while only the mutated allele was detected in clone a2. Employing a calibration curve obtained from the Delta Rn values for the wild-type (WT) and mutated (M) alleles of different proportions of al and a2 cell mixtures, the percentage of a2-like cells (only M allele, i.e. double-mutant) for different tissues were determined. The proportion of double-mutant, a2-like cells was higher in ossified tissues, such as muscle, and tissues adjacent to ectopic bone, such as fat and fibrous tissue. The highest percentage of a2-like cells was found in nervous tissues. Surprisingly, certain tissues, including healthy skin, had a Delta Rn ratio higher than al cells, suggesting the presence of cells with only WT allele (Figure IE).

[0161] Example 2.2. Biallelic inactivation of GNAS increases the expression of bone and cartilage-related genes

[0162] In addition to differing expression levels of GNAS, clones al and a2 showed differential expression of genes associated with the process of ectopic bone formation. The expression of leukaemia inhibitory factor (LIF), an inductor of ossification, was almost twenty times higher in a2 cells. The expression of SRY-Related HMG Box Gene 9 (SOX9), a key transcription factor for chondrogenesis, and aggrecan (ACAN), one of the main components of cartilage extracellular matrix, was also higher in a2 cells. The same trend was followed by BMP2, an activator of osteogenic genes; chemokine (C-C motif) ligand 2 (CCL2), a monocyte chemoattractant protein; hypoxia-inducible factor (HIF), an inductor of angiogenesis and cartilage differentiation; uncoupling protein 1 (UCP1), expressed by ectopic bone-associated brown adipocyte-like cells; and TGFpi, an inductor of ectopic bone formation (Figure 2A).

[0163] Clone al showed higher expression of the bone-related transcription factor Runt-related transcription factor 2 (RUNX2) under osteoblastogenic and adipogenic stimuli (Figure 2B-C), while clone a2 showed a significant upregulation of its expression under chondrogenic stimuli (Figure 2D). Interestingly, even though there was no difference in SPP1 expression at basal conditions, clone a2 showed a notable upregulation under all the differentiation stimuli,especially during chondrogenesis (Figure 2B-D). Strikingly, SPP1 was downregulated in clone al but upregulated in clone a2 during adipogenesis (Figure 2C). Alkaline phosphatase (ALPL) expression was also higher in a2 cells under all three conditions, with the most notable differences found upon chondrogenic induction (Figure 2B-D). The expression of the cartilage marker aggrecan (ACAN) was also significantly higher in clone a2 in all three conditions, and its expression was induced in clone a2 under all three stimuli (Figure 2B-D).

[0164] Moreover, during chondrogenesis, SOX9 expression was induced in clone a2, but not in clone al. Unexpectedly, type I collagen (COL1) was also upregulated in clone a2 during chondrogenesis. Type II collagen (COL2) was upregultaed only in clone a2 during the same process, while type X collagen was upregulated in both clones (COL 10) (Figure 2D).

[0165] Example 2.3. GNAS double-mutant cells inhibit adipogenesis, induce osteochondrogenesis and promote inflammation through paracrine signalling

[0166] Through cell differentiation experiments in wild type cell lines, the secretome of a2 cells was demonstrated to have adipogenesis-inhibiting and chondrogenesis-inducing activity. After 7 days of adipogenic differentiation, all the adipogenesis-related genes studied (peroxisome proliferator-activated receptor y (PPARG), fatty acid-binding protein 4 (FABP4), adiponectin (ADIPOQ), and perilipin 2 (PLIN2)) were upregulated in C3H10T1 / 2 cells; however, a2 cells secretome reduced the induction of all four genes, unlike al cells secretome (Figure 3 A). On ATDC5 cells subjected to 7-day chondrogenic induction, a2 cells secretome induced the expression of SOX9 and ACAN but reduced the expression of COL2. The expression of collagenase 3 (MMP13) was also significantly induced (Figure 3B). In addition, a2 cells secretome significantly induced the expression of SSP1 and transforming growth factor P-induced (TGFBi), two factors closely related to HO pathogenesis, in both adipogenically- and chondrogenically-induced cells (Figure 3A-B).

[0167] On the osteoblastic cell line SaOS-2, a2 cells secretome also induced the expression of SPP1, as well as that of the chemotactic factors CCL2 and vascular cell adhesion molecule (VCAM), while the expression of the transcription factors RUNX2 and SOX9 remained unchanged (Figure 3C). In co-culture experiments, a2 cells induced the expression of SPP1, CCL2, VCAM, ICAM, IL-ip, and TGFpi in THP-1 monocytes. While al cells also induced the expression of some of these genes, all of them related to the pathogenic ossification process, the upregulation of the key factors SPP1, CCL2, and TGFpi in monocytes cocultured with a2 cells were significantly higher (Figure 3D). Overall, these resultsdemonstrate the ability of a2 cell secretome to induce the expression of SPP1 and activate TGFpi signalling, both of which are key factors in ectopic bone formation, across a broad range of cell types.

[0168] In order to identify which protein(s) were responsible for the a2 paracrine effects, the same experiments were performed using the secretome of a2 cells divided into fractions of decreasing molecular weights. Interestingly, only the fraction containing proteins larger than 100 kDa showed the same adipogenesis-inhibiting activity as the whole secretome (Figure 4A). Furthermore, this protein fraction exhibited a chondrogenesis-inducing effect similar to that of the whole secretome and retained the ability to induce the expression of SPP1 and TGFBi under both adipogenic and chondrogenic stimulation (Figure 4A-B).

[0169] Example 2.4. Silencing of GNAS inhibits adipogenesis and induces osteochondrogenesis

[0170] GNAS silencing in the mesenchymal progenitor cell line C3H10T1 / 2 resulted in enhanced SPP1 expression after 7 days of either osteoblastogenic or adipogenic differentiation, even though GNAS silencing was not sustained during osteoblastogenesis (Figure 5A). During adipogenesis, GNAS silencing impaired the formation of lipid droplets (Figure 5B) and increased the expression of the cartilage- and bone-related transcription factors SOX9 and RUNX2, as well as the main collagen of growth plate cartilage, COL2 (Figure 5C).

[0171] Example 2.5. GNAS single- and double-mutant cells show widespread proteomic divergence

[0172] A total of 1,185 proteins were detected in al and a2 clones by SWATH mass spectrometry, out of which 685 (58%) were differentially expressed; 336 proteins were upregulated in a2 cells, while the other 349 were downregulated. The ten most upregulated proteins in a2 cells compared to al (FC > 4, p < 0.05) were eukaryotic translation initiation factor (EIF6), a-fetoprotein (AFP), copine-1 (CPNE1), poliovirus receptor (CD155), ATP-dependent RNA helicase DDX19B, ATP-dependent DNA helicase QI (RECQL), CTP synthase 1 (CTPS1), ATP-dependent 6-phosphofructokinase (PFKP), UDP -glucose 6-dehydrogenase (UGDH), and ATP-dependent RNA helicase DDX27. The ten most downregulated proteins (FC < 0.3, p < 0.05) were receptor expression-enhancing protein 5 (REEP5), cathepsin Z (CTSZ), protein S100-A4 (S100A4), protein NDRG1, BTB / POZ domain-containing protein KCTD12, galectin-3 (LGALS3), superkiller complex protein 3 (SKIC3), cathepsin B (CTSB), protein S100-A6 (S100A6), and heat shock protein beta-1 (HSPB1). In contrast, DDA proteomicanalysis of the >100 kDa fraction of al and a2 secretomes identified only 42 proteins, with a-2 -macroglobulin (A2M) being the only one significantly up-regulated in a2 cells. Other pro-osteoblastogenic factors present in a2 secretome included apolipoprotein- A, COL1, periostin, vitronectin, and complement molecules C3 and C5.

[0173] Example 2.6. Transcriptomic analysis of double-mutant cells reveals enrichment of genes related to bone formation and inflammation

[0174] RNA-seq analysis of al and a2 cells identified 5,189 differentially expressed genes (2,618 upregulated and 2,571 downregulated), with a threshold of ±0.3 log2 fold change and a p-value cut-off of 0.05. Consistent with our previous findings and the phenotypic differences observed between the two cell lines, genes related to bone formation (BMP2, LIF), cartilage (ACAN), and chemotaxis (CCL2) were upregulated in a2 cells. Other upregulated genes included matrix degrading enzymes related to bone metastasis (MMP-1), endochondral ossification (MMP-10), and adipogenesis inhibition (ADAMTS18), as well as chemokines CCL7, CXCL9, CXCL5, and CXCL11. Interestingly, one of the most upregulated genes was the transcription factor SOX11, whose expression is significantly induced in adult neurons in response to injury. The transcription factor RUNX3, which is activated by TGFP and BMP signalling and promotes chondrocyte maturation during osteochondral ossification, was also highly upregulated. In contrast, transcription factors associated with adipogenesis, such as PPARy, C / EBPa, C / EBP5, and KLF5, were downregulated in a2 cells. As expected, GNAS was also markedly downregulated. Similarly, KEGG pathway enrichment analysis revealed significant downregulation of cAMP signalling and adipocyte lipolysis, while inflammatory pathways such as chemokines, TLRs, TNF and IL- 17 signalling were upregulated. Enrichment analysis using the ARCHS4 Tissues database showed that the top enriched term for a2 cells was “astrocyte” (adjusted p-value = 5.328 x 1 O’7, combined score = 27.26), while for al cells, it was “fibroblast” (adjusted p-value = 3.089 x 10'58, combined score = 347.77).

[0175] Example 2.7. Self-amplifying mRNA transfection restores the expression of GNAS

[0176] The transfection with GNAS samRNA restored GNAS expression in a2, double-mutant cells, after 24 hours of treatment (Figure 6).

Claims

CLAIMS1. In vitro method for the diagnosis and / or prognosis of heterotopic ossification comprising identifying cells characterized by the biallelic inactivation of GNAS in ectopic bone tissue and / or surrounding tissues obtained from a subject, wherein the presence of said cells indicates that the subject may be suffering from genetic heterotopic ossification.

2. Method for screening compounds for use in a method for the treatment and / or prevention of heterotopic ossification which comprises assessing whether the candidate compound is capable of reducing the amount or eliminate the cells characterized by the biallelic inactivation of GNAS in ectopic bone tissue and / or surrounding tissues, wherein the reduction or elimination of said cells is indicative that the compound may be used for the treatment and / or prevention of heterotopic ossification .

3. Cell line, or secretome derived thereof, wherein the cell line is characterized by the biallelic inactivation of GNAS, and wherein said cell line is specific to the ectopic bone tissue and / or surrounding tissues.

4. A method for selecting a treatment for heterotopic ossification, comprising determining the presence of cells with biallelic inactivation of GNAS in ectopic bone tissue and / or surrounding tissues, wherein the presence of said cells is indicative of a specific phenotype of heterotopic ossification and can guide the selection of an appropriate therapeutic approach.

5. Cell suspension or cell population comprising the cells according to claim 3, or the secretome derived thereof.

6. A composition comprising the cell suspension or cell population of claim 5, or the secretome derived thereof, for use in a method for promoting osteogenesis, for promoting chondrogenesis and / or for inhibiting adipogenesis.

7. Composition for use, according to claim 6, in a method for the treatment and / or prevention of a disease which may benefit from promoting the osteogenesis selected from: Osteoporosis, Osteogenesis Imperfecta (01), Fracture Healing and Bone Defects, Paget’s Disease of Bone, Osteomalacia and Rickets, Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), Osteonecrosis (Avascular Necrosis), Bone Loss from Prolonged Immobilization, Metabolic Bone Diseases, Bone Loss Due to Hormonal Changes and / or Bone Grafts and Orthopedic Surgeries;and / or for the treatment and / or prevention of diseases which may benefit from promoting chondrogenesis selected from: Osteoarthritis, rheumatoid Arthritis, traumatic cartilage injuries, intervertebral disc degeneration, chondrodysplasias, osteochondritis dissecans, avascular necrosis, temporomandibular joint disorders, congenital cartilage defects and / or post-surgical cartilage loss, and / or for the treatment and / or prevention of a disease which may benefit from inhibiting adipogenesis selected from obesity and metabolic syndrome, type 2 diabetes, cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), cancer or inflammatory diseases.

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